Genomic characterisation of possible symbiotic pathogenic bacteria species isolated from undescribed Cruznema nematodes bacterivore

Abstract

Pest infestation has devastating effects towards food security and gross domestic product (GDP) of the country. The use of synthetic chemical pesticides is common throughout the world, and it believe to be an ultimate solution for pest attacks on farms. However, the long use of these synthetic pesticides has led to environmental and human health hazard. Entomopathogenic nematodes (EPNs) are microscopic insect parasitic worms that are found in different ecological niche and classified as biological agent. EPNs have symbiotic relationship with pathogenic bacteria from a phylum of Pseudomonadota, a class of Gammaproteobacteriar. These bacteria synthesize bio-active toxic compounds which are responsible for killing the insect host. Cruznema nematodes belong to the phylum order Rhabditida as EPNs. This study explores the possible symbionts bacteria of Cruznema sp. NTM 2021 and their genomic make-up. Molecular identification of Cruznema nematodes was performed, which involves DNA extraction, PCR, DNA purification and 18S rDNA sanger sequencing. Furthermore, Cruznema sp. NTM 2021 gut microbiome was explored through metagenomics sequencing using PacBio sequencer and the results showed that Pseudomonadales dominated with 40 %, Hyphomicrobiales 23 % and Bacteroidetes 22 %. Moreover, two novel bacteria (namely Enterobacter sp. PTB and Pseudomonas sp. PKS) were isolated from Cruznema sp. NTM 2021 and pass a symbiosis test for Cruznema sp. NTM 2021. Both Enterobacter sp. PTB and Pseudomonas sp. PKS Cruznema bacteria symbionts were assessed for their pathogenic against Tenebrio Molitor (T. molitor), and both bacteria were found to be pathogenic. Furthermore, both bacteria were found to produce metabolites. The metabolites were checked for their effectiveness as antimicrobial agents against common antibiotic resistant bacteria (namely Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus). The metabolites produce by both bacteria were found to be bacteriostatic against all four antibiotic resistant bacteria. The metabolites identification of both Cruznema bacteria symbionts was performed using high pressure liquid chromatography (HPLC). Further five metabolites were found to be produced by Enterobacter sp. PTB meanwhile 15 metabolites were identified to be produced by Pseudomonas sp. PKS. Whole genome sequencing and annotation of both Enterobacter sp. PTB and Pseudomonas sp. PKS was achieved through sequencing the genomic DNA of each bacterium separately by Illumina Miseq. The quality control was conducted with the aid of FastQC and Trimmomatric. The assembling of genomic reads was conducted using de novo method on SPADES tool and annotation was conducted using RAST tool. Enterobacter sp. PTB genome is comprise of 5 609 562 bp, 54.2 % GC content, 76 RNAs, 572 subsystems whereas Pseudomonas sp. PKS genome is comprise of 5 660 896 bp, 54.2 % GC content, 75 RNAs, 569 subsystems. However, Antismash tool reveals that both bacteria species consist of 5 secondary metabolites gene cluster which produce compound such as NI-siderophore, Thiopeptide, Arylpolyne, NRPmetallophore, Hserlactone.